Rgg-associated SHP signaling peptides mediate cross-talk in Streptococci

Abstract

We described a quorum-sensing mechanism in the streptococci genus involving a short hydrophobic peptide (SHP), which acts as a pheromone, and a transcriptional regulator belonging to the Rgg family. The shp/rgg genes, found in nearly all streptococcal genomes and in several copies in some, have been classified into three groups. We used a genetic approach to evaluate the functionality of the SHP/Rgg quorum-sensing mechanism, encoded by three selected shp/rgg loci, in pathogenic and non-pathogenic streptococci. We characterized the mature form of each SHP pheromone by mass-spectrometry. We produced synthetic peptides corresponding to these mature forms, and used them to study functional complementation and cross-talk between these different SHP/Rgg systems. We demonstrate that a SHP pheromone of one system can influence the activity of a different system. Interestingly, this does not seem to be dependent on the SHP/Rgg group and cross-talk between pathogenic and non-pathogenic streptococci is observed.

Figure 1. Description of strains containing P_shp-luxAB transcriptional fusions in various genetic backgrounds.

These strains were constructed in S. thermophilus strain LMD-9 and used to study the expression of the shp genes of S. agalactiae strain NEM316 (shp/gbs1555 locus) and S. mutans strain UA159 (shp/SMU.1509 locus) in the presence and absence of the corresponding shp and rgg genes and in the presence and absence of the ami genes of S. thermophilus strain LMD-9.

Figure 2. Growth and luciferase activities of strains containing P_shp-luxAB fusions in various genetic backgrounds.

Growth curves (OD₆₀₀) are presented in gray and relative luciferase activities (RLU/OD₆₀₀) in black. Growth and relative luciferase activities of derivatives of S. thermophilus strain LMD-9 grown in CDM and containing P_shp-luxAB fusions of the loci shp/gbs1555 of S. agalactiae (A), shp/SMU.1509 of S. mutans (B) and shp/ster_1299 of S. thermophilus strain LMD-9 (C). The genetic backgrounds are indicated as follows: (•) the shp and rgg genes of the locus tested and the ami gene of S. thermophilus are present (▴) the cognate shp gene of the locus studied is not present, (▪) the cognate rgg gene of the locus studied is not present and, (×) the ami genes of S. thermophilus are not present. Experiments were done at 30°C for the shp/gbs1555 and the shp/SMU.1509 loci and at 42°C for the shp/ster_1299 locus. Data shown are representative of three independent experiments.

Figure 3. Fragmentation spectra of the ions of mature forms of SHP1299, SHP1555 and SHP1509.

Fragmentation of the ions m/z 1018.56 (A) and m/z 564.28 (B) identified in the supernatant of cultures of S. thermophilus strain LMD-9. Fragmentation of the ions m/z 799.49 (C) identified in the supernatant of cultures of S. agalactiae strain NEM316 and m/z 872.5 (D) identified in the supernatant of cultures of S. mutans strain UA159. All ions were analyzed in the linear ion trap.

Figure 4. Cross-complementation of the shp/rgg loci with synthetic SHP pheromones.

Maximum relative luciferase activities of the reporter strains TIL1052 (shp1299::erm blp::P_shp1299-luxAB aphA3), TIL1200 (Δshp1358 blp::P_shp1358-luxAB), TIL1382 (blp::gbs1555::P_shp1555-luxAB aphA3) and TIL1384 (blp::SMU.1509::P_shp1509-luxAB aphA3) grown in the absence (grey) or in the presence of synthetic SHP peptides added at the beginning of the culture to a concentration of 1 µM: EGIIVIVVG (green), DILIIVGG (red), DIIIIVGG (blue), ETIIIIGGG (purple), DIIIFPPFG (yellow). The legitimate SHP synthetic peptide associated to the locus studied is hatched in each case.